Membrane fusion provides a conceptually simple mechanism for enveloped viruses to deliver their genomes into the cytoplasm of target cells. In contrast, the mechanisms used by nonenveloped viruses to penetrate cellular membranes and release their genomes into the cell remain poorly understood. This application proposes a program of structural, biochemical and biophysical studies of poliovirus cell entry intermediates as a simple model for understanding how nonenveloped viruses gain entry into the cell. The problem is framed in the context of several specific questions: 1) What is the nature of the virus-receptor interaction and how is receptor binding coupled to the induction of conformational changes? 2) What are the sequence of conformational changes that ultimately leads to cell entry and the release of the viral RNA? 3) What is the nature of the interaction of the virus with the cell membrane that leads to the internalization of the RNA? 4) How is the RNA released from the particle, across a vesicle membrane and into the cell? The central hypotheses governing this work are: that the machinery for this process with poliovirus will be directly applicable to closely related viruses (many of which including enterovirus 71 (EV71) and Coxsackievirus A16 (CAV16) are significant human pathogens): that some of features of the machinery will be conserved in more distantly related viruses, and that the nature of the machinery used by viruses will be relevant to understanding how nucleic acids and other large cargoes are transported across membranes in cells From a structural perspective, these questions cover over six orders of magnitude in scale from the atomic level (tenths of nanometers) to the cellular level (tens of microns). This range is well beyond the scope of any one structural method. The project seeks to develop a hybrid approach combining x-ray crystallography, cryoelectron microscopy, and cryoelectron tomography to develop a series of structural snapshots of cell entry intermediates of poliovirus; and to use these structures to address questions posed by, and pose questions for, supporting biochemical and genetic studies.
The specific aims i nclude structural studies of soluble cell entry intermediates in vitro t near-atomic resolution and of membrane-associated poliovirus cell entry intermediates in vitro at nanometer resolution.
The aims also include the use of a simple receptor-decorated liposome model to identify the constituents of the channel that supports release of the genome across membranes and to characterize the kinetics and mechanism of RNA translocation at the single particle/single liposome level. The goal will be to define structural changes associated with cell entry and delivery of the genome into the cytoplasm at an unprecedented level of detail. The results are expected to be relevant to the design of novel antivirals and thermostable vaccines against poliovirus and related viruses including EV71 and CAV16.

Public Health Relevance

The project seeks to understand how poliovirus and related viruses such as rhinoviruses, coxsackieviruses, and enteroviruses (all of which are significant human pathogens) enter cells. The findings are expected to be more generally relevant to many other viruses that lack a lipid envelope and to cellular membrane transport mechanisms. Understanding structural changes required for infection could lead to the development of novel antivirals and thermostable vaccines.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Study Section
Virology - A Study Section (VIRA)
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Park, Eun-Chung
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Harvard Medical School
Schools of Medicine
United States
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Zhao, Zhao; Zhang, Meng; Hogle, James M et al. (2018) DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry. J Am Chem Soc 140:10639-10643
Nasr, Mahmoud L; Baptista, Diego; Strauss, Mike et al. (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14:49-52
Groppelli, Elisabetta; Levy, Hazel C; Sun, Eileen et al. (2017) Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes. PLoS Pathog 13:e1006197
Strauss, Mike; Schotte, Lise; Karunatilaka, Krishanthi S et al. (2017) Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State. J Virol 91:
Strauss, Mike; Schotte, Lise; Thys, Bert et al. (2016) Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site. J Virol 90:3496-505
Schotte, Lise; Thys, Bert; Strauss, Mike et al. (2015) Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs). Antimicrob Agents Chemother 59:4695-706
Strauss, Mike; Filman, David J; Belnap, David M et al. (2015) Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry. J Virol 89:4143-57
Schotte, Lise; Strauss, Mike; Thys, Bert et al. (2014) Mechanism of action and capsid-stabilizing properties of VHHs with an in vitro antipolioviral activity. J Virol 88:4403-13
Butan, Carmen; Filman, David J; Hogle, James M (2014) Cryo-electron microscopy reconstruction shows poliovirus 135S particles poised for membrane interaction and RNA release. J Virol 88:1758-70
Panjwani, Anusha; Strauss, Mike; Gold, Sarah et al. (2014) Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore. PLoS Pathog 10:e1004294

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